Experiment9-Plastid - Experiment 9(Lab period 10 Cell...

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Experiment 9 (Lab period 10) Cell fractionation and isolation of chloroplasts The green color of the leaves, and sometimes the stems, of plants is due to the presence of the green pigments chlorophyll a and chlorophyll b in the subcellular organelles called chloroplasts. The remainder of a green cell is typically colorless. The green organelles lie free in the cytoplasm of the cell, unattached to other cellular components such as the wall, the membranes, the nucleus, and the mitochondria. When the cell wall is disrupted, the cell membrane breaks, and the subcellular components are released as separate particles of various sizes and densities. In this laboratory, the cells of spinach leaves will be disrupted, freeing the untethered organelles, which can then be sorted out from each other by filtration and differential centrifugation as cell fractions. Filtration will remove large debris (e.g., cell walls) and unbroken cells, providing a filtrate that contains organelles (nuclei, chloroplasts, mitochondria, ribosomes), small membrane vesicles, and soluble components; most of these will not be visible in the light microscope. Low-speed centrifugation will sediment remaining large bodies from the filtrate, and moderate-speed centrifugation will sediment chloroplasts, leaving mitochondria and ribosomes and soluble components in the supernatant. (The mitochondrial fraction could be collected by high-speed, and ribosomal and membrane vesicle fractions by ultra-high-speed centrifugation.) Repeated rounds of differential centrifugation can be used to further purify the chloroplasts when highly purified preparations are required for experimentation, but one round of low-then-moderate centrifugation will suffice for the purposes of this exercise. In this laboratory, you will prepare a crude suspension of chloroplasts and determine several
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This note was uploaded on 01/30/2012 for the course BIO 2004 taught by Professor Morton during the Spring '11 term at Columbia.

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Experiment9-Plastid - Experiment 9(Lab period 10 Cell...

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